Recombinant or synthetically produced peptides have important applications as pharmaceuticals. Peptides, however, often suffer from poor metabolic stability, poor cell penetrability, and promiscuous binding due to conformational flexibility. One approach to stabilizing these peptides is to use intramolecular crosslinkers to maintain the peptide in the desired configuration, for example using disulfide bonds, amide bonds, or carbon-carbon bonds to link amino acid side chains. See, e.g., Jackson et al. (1991), J. Am. Chem. Soc. 113:9391-9392; Phelan et al. (1997), J. Am. Chem. Soc. 119:455-460; Taylor (2002), Biopolymers 66: 49-75; Brunel et al. (2005), Chem. Commun. (20):2552-2554; Hiroshige et al. (1995), J. Am. Chem. Soc. 117: 11590-11591; Blackwell et al. (1998), Angew. Chem. Int. Ed. 37:3281-3284; Schafmeister et al. (2000), J. Am. Chem. Soc. 122:5891-5892; Walensky et al. (2004), Science 305:1466-1470; Bernal et al. (2007), J. Am. Chem Soc. 129:2456-2457; U.S. Pat. No. 7,192,713 B1 (Verdine et al) (describing cross-linked stabilized-helical peptides comprising natural and non-natural amino acids, wherein the peptide comprises at least two reactive moieties capable of undergoing a C—C bond-forming reaction); and U.S. Pat. No. 5,811,515 (Grubbs et al) (describing the synthesis of conformationally-restricted/cyclic-stabilized peptides and peptidomimetics from precursors containing two or more unsaturated C—C bonds); the contents of which patents and publications are incorporated herein by reference. Such polypeptides which are conformationally stabilized by means of intramolecular cross-linkers are sometimes referred to as “stapled” polypeptides.
A major advantage of these crosslinked polypeptides is that they have an enhanced ability to penetrate cell membranes relative to their non-stapled counterparts. This cellular uptake is believed to be mediated by an active transport mechanism utilizing endocytosis.
Some of the physical characteristics which facilitate the entry of the peptides into the cells also tend to increase the affinity of crosslinked peptides to serum proteins, such as albumin. Consequently, many highly promising leads exhibit a marked “serum shift”, having greatly diminished activity in vivo or in assays having serum based media, compared to activity in assays using serum-free media, rendering the peptides less than optimal for therapeutic or diagnostic applications. Crosslinked polypeptides having low levels of serum binding, however, tend to have poor cell penetration, as well as poor pharmacokinetics, e.g., rapid renal or first pass clearance. This invention addresses this and other problems.